Abstract

Site-saturation mutagenesis has been used to substitute lysine 234 in RTEM-1 [beta]-lactamase with 18 natural amino acids. Lysine 234 is conserved among the class A [beta]-lactamases and has been implicated in x-ray diffraction and molecular modeling studies as a cationic residue that binds the C3 carboxylate common to most [beta]-lactamase substrates (Hertzberg and Moult (1987) Science, 236, 694.; Meows et al. (1990) PROTEINS: Structure, Function, and Genetics, 7, 156.; Chapter 2 of this Thesis).

The 234 mutant proteins have significantly reduced activity towards hydrolysis of penicillin, ampicillin, and cephalosporin C and do not have observable phenotypic activity towards hydrolysis of cephalothin. With the exception of K234R, the phenotypic activities are essentially indistinguishable. The high phenotypic resistance of the K234R mutant shows that a positively charged guanidinium group can effectively substitute for the wild-type ammonium group of lysine 234. The uniformly reduced phenotypic activities of all other mutants indicate that the mutant amino acids do not interfere with or participate directly in the enzymatic reaction mechanism. Behavior of the mutants on anion exchange columns and analysis by circular dichroism spectroscopy show that the secondary and tertiary structures characteristic of the wild-type enzyme are not significantly disrupted by mutation.

Four mutants were purified to homogeneity and the steady-state kinetic parameters for hydrolysis of benzylpenicillin were measured for each. The kinetics show that substituting lysine 234 with glutamate, glutamine, or valine causes a decrease in the apparent equilibrium-binding constant for benzylpenicillin of about three orders of magnitude. In contrast, the binding constant for the K234R mutant, which preserves the positive charge at position 234, is reduced by only one order of magnitude. These results indicate that lysine 234 is responsible for stabilizing the RTEM-1/benzylpenicillin complexes by approximately 4.5 kcal mol(-1), consistent with the formation of an intermolecular charged hydrogen bond.

The mutations have much less pronounced effects on the ability of the enzyme to catalyze the hydrolysis of benzylpenicillin under saturating substrate concentrations. K234R and K234E have the wild-type kcat, while K234Q and K234V have kcats reduced by one and two orders of magnitude, respectively.

Analysis of the kinetic results shows that lysine 234 performs a uniform binding function in catalysis by RTEM-1 lactamase (Albery and Knowles (1976) Biochemistry, 15, 5631). This contrasts with the differential binding role that has been determined for lysine 234 in catalysis by [beta]-lactamase from Bacillus licheniformis (BL) (Ellerby et al. (1990), Biochemistry, 29, 5797). The results indicate that RTEM-1 utilizes the binding capabilities of lysine 234 to increase the catalytic rate under low substrate concentrations, while BL utilizes it to increase the rate under conditions of high substrate concentration.

In Chapter 2, molecular modeling of the RTEM-1 [beta]-lactamase active site is described. The models were based on the published [alpha]-carbon trace of [beta]-lactamase from Staphylococcus aureus PC1 (Hertzberg and Moult, ibid.) and were built in the absence of an experimentally determined high-resolution x-ray structure of a class A [beta]-lactamase. The experiments were designed to yield structures useful for rationalizing the results of mutagenesis experiments and for formulating mechanistic ideas testable by experiment. The model structures are shown to compare favorably with the high-resolution structure of a class A [beta]-lactamase published after the modeling was completed. Two working mechanisms for catalysis by RTEM-1 are proposed, based on the modeling, mutagenesis results, and on precedent.

Chapter four describes the development of methods for measuring enzymatic reaction rates with circular dichroism spectroscopy (CD). Application of the methods to two enzymes, [beta]-lactamase and ribulose-1,5-bisphosphate carboxylase/oxygenase, is described. The general utility of the methods is addressed.